Abstract A bottleneck in drug discovery is the lack of high-quality small-molecule probes to validate the disease relevance of genes, particularly those with promising disease linkage but are considered ?undruggable?, such as transcription factors and scaffolding proteins. Recently, covalent drugs have become a promising option to address a number of challenges such as low efficiency of lead discovery, resistance to targeted therapies, and poor druggability of many targets. We hypothesize that the druggable targets can be dramatically expanded by leveraging covalent chemistry in drug development. The TarGead Sciences team proposes to combine bio-orthogonal reactions with MS- based proteomics technology to create a novel platform that allows for the detection and quantification of the interactions between an electrophilic compound and thousands of proteins across the proteome. Specifically, we will synthesize a library of electrophilic probes based on privileged scaffolds as baits to fish out novel targets in various cell types (Aim 1). A new chemical proteomic approach will be established to map and quantify the proteome-wide interactions of electrophilic probes in different cell lines (Aim 2). In Aim 3, we will optimize electrophilic leads for potent and selective functional modulation of select targets such as vacuolar ATPase. Our work will yield high-quality covalent leads for novel targets including the undruggable ones, paving the way for subsequent preclinical studies.